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Alternative Way of Staining a Gel
FOR LABELING GELS, PLEASE SEE THE LABELING GELS ARTICLE. Follow the guidelines in this article for all gel labeling. Alternate Way of Running and Staining a Gel Before beginning this protocol, make sure that you keep track of which sample is going in which lane early on. I found that writing this down early makes it less likely that you mess up during the loading process. What I like to do is make a table on my notebook that looks just like the gel, and I write down which sample is supposed to go in which lane. 1. Following the same protocol listed in the page before when pouring the gel, except DO NOT add EtBr into the gel before it solidifies. 2. While waiting, you can add the loading dye to your samples. So far, we have been adding a 1/5 of 6X loading dye for every µL of your sample. So if you have 25 µL of sample in each PCR tube, each tube would receive 5 µL of loading dye. 3. We now need to create a ladder in order to compare band sizes. The ladder (which is µL in volume) is typically placed in just one end of the well, but for safekeeping, we usually load it on both ends of the gel. Like step 2, 6X loading dye is also added to the ladder. The difference is that we need 1/6 of loading dye for 1 µL of the ladder. So if we want two ladders, we need to add 0.33 µL of dye to an extra PCR well. 4. By this time, your gel should have solidified. After pouring 1X TAE buffer (located on Chen Yu's bench) on your gel box, you are now ready to load your samples in the gel. The first thing I do is load the 1 µL of the ladders first on the ends of my gel, that way I don't lose track of it while loading my samples. Then, pipet out 10 µL of your sample and load it to each well. Lots of people have their own method of loading the samples in the gel, but the one that works best for me is Mark's method of using the back of your fingers to stabilize the pipette tip. To make sure that your tip is actually inside the gel, you can carefully wiggle the tip around. If the gel moves, then you're in. When loading the sample, it is crucial that you don't pipet the samples all the way through because this could potentially cause bubbles. What we do is pipet until the reach where the pipet stops the first time, and pull the pipet out of the well and discard. 5. You can now cover up the gel box with its appropriate cover, hook up the cables correctly (red to red, black to black) and turn on the kit. We found that using a voltage between 100-120V works best. More specifically: about 10V/cm , where cm is the length of the box in centimeters, is best- for us that's about 160V for the big boxes and 100V for the small ones. Grab a timer and set it for about 30-40 minutes. Look at your gel periodically to make sure that samples haven't gone a length of more than 3/4 of the gel. 6. Once 30 minutes have passed, you can now unplug all the cables and carefully transfer the gel into some kind of container. Mark has instructed me to use the glass pyrex container with the blue lid which is typically just placed on one of the racks around the lab. Basically glass is better than plastic for this, plastic will absorb the EtBr. Transfer some of the TAE buffer from your gel box to the container with the gel, filling up just enough to cover the height of the gel. 7. Then, using gloves, grab the EtBr from the chemical rack and pipet 6 µL on opposite sides of the container. Place the lid on the container quickly after dispensing since EtBr is light-sensitive. Place the container in a shaker for Western blots, and set the RPM at a low speed, just enough for the EtBr to spread throughout the gel. Grab your timer again and wait 40 minutes. The tips used to pipet EtBr need to go in the Ethidium Bromide solid waste bucket under the gel bench. 8. After 40 minutes, discard all the TAE buffer in the liquid EtBr waste container under the hood. Then add the same volume of H2O, just enough to fill the height of the gel, and have it shake for 10 more minutes. 9. Discard the H2O in the liquid EtBr waste container and grab the Saran wrap from either Katie or Shih Yin's desk and head to the gel camera upstairs. 10. Login the computer upstairs using your Buck username and password and run the only program that doesn't fit in. This is the program you use to run the gel camera. If you've never taken a picture before, this should probably be shown to you the first time. After you have taken a good picture of your gel, email this to yourself and discard your gel into the solid EtBr waste container. Email yourself the .scn file of the software, the EXPRT RAW IMAGE AS TIFF pic, and the EXPORT DISPLAYED IMAGE AS JPG pic. 11. You can now analyze your gel using the picture you took. It's best to open the jpg in GIMP, crop it to just the gel, and do image>mode>grayscale followed by colors>levels>auto, then save and print this, and label the ladder sizes and what is in each lane etc.